ORIGINAL ARTICLE Sequence-Dependent Synergy Of

Sequence-dependent synergy of the proteasome inhibitor bortezomib and cytarabine in mantle cell lymphoma

O Weigert1,2,3, A Pastore1,2,3, M Rieken2, N Lang1, W Hiddemann1 and M Dreyling1,2

1Department of Internal Medicine III, University of Munich, University Hospital Grosshadern, Munich, Germany and 2GSF-National Research Center for Environment and Health, Haematologicum, Munich, Germany

Single-agent bortezomib, a potent, selective and reversible consisted of CHOP (cyclophosphamide, vincristine, doxorubi- inhibitor of the 26S proteasome, has demonstrated clinical cin and prednisone),12 R-FCM (rituximab, fludarabine, cyclo- efficacy in relapsed and refractory mantle cell lymphoma (MCL). 13 Objective response is achieved in up to 45% of the patients; phosphamide, and mitoxantrone) and gemcitabine/oxaliplatin however, complete remission rates are low and duration of with all relapses occurring within 1 year. No response was seen 90 response proved to be short. These limitations may be after radioimmunotherapy with rituximab/ Y-ibritumomab– overcome by combining proteasome inhibition with conven- tiuxetan 5 months earlier. Salvage therapy was started with tional chemotherapy. Here we present two case reports and in single-agent bortezomib 1.3 mg/m2 given intravenously twice vitro data suggesting synergistic efficacy of bortezomib weekly for 2 consecutive weeks, followed by a 1-week rest combined with cytarabine in MCL. Interestingly, efficacy in vitro correlated with sequence of treatment, indicating that period. Because of progressive lymphadenopathy at week 5, pretreatment with cytarabine, followed by proteasome inhibi- treatment was changed to a combination regimen consisting 2 tion, may be the preferred approach. of bortezomib (1.5 mg/m , days 1 and 4) and cytarabine Leukemia (2007) 21, 524–528. doi:10.1038/sj.leu.2404511; (1000 mg/m2 as a 3-h infusion on days 2 and 3) applied in published online 1 February 2007 3-week intervals. After two cycles, the leg edema and Keywords: non-Hodgkin’s lymphoma; mantle cell lymphoma; lymphadenopathy improved dramatically, and a very good bortezomib; cytarabine; combination treatment partial response was confirmed by computer tomography (CT) scanning (Figure 1a and b). Because of NCI/CTC grade 4 neutropenia after cycle 2, the dose of cytarabine was reduced to 75% and granulocyte-colony-stimulating factor (G-CSF) support was initiated. Importantly, there was no infectious complication Introduction and only cycle 4 had to be delayed for 1 week. Because of NCI/ CTC grade 3 sensory peripheral neuropathy after cycle 2, The ubiquitin–proteasome pathway is essential for maintaining bortezomib was omitted during the subsequent two cycles with intracellular protein homeostasis and represents a valid target for polyneuropathy gradually improving; the hematologic toxicity the treatment of malignant disease.1 Various oncogenes and did not differ significantly after bortezomib was held. Except for regulatory proteins for cell cycle progression and apoptosis are alopecia, there was no additional significant non-hematological processed by this pathway.2 Bortezomib is a potent, selective toxicity. Meanwhile, 11 months (343 days) after initiation of and reversible inhibitor of the proteasome with demonstrated combination therapy, the patient suffered from systemic progres- efficacy in relapsed multiple myeloma.3,4 Single-agent bortezo- sion and was started on a bendamustin-containing regimen. mib demonstrated clinical activity in several other hematologic malignancies5 with especially encouraging results in relapsed or 6–8 refractory mantle cell lymphoma (MCL). Objective response Case report 2 is achieved in 29–45% of MCL patients; however, complete A 63-year-old woman with relapsed stage IV MCL presented remission rates are low and median duration of response short. with progressive abdominal lymphadenopathy and reappear- These limitations may be overcome by combining bortezomib ance of extensive watery diarrhea, attributable to exacerbation 9,10 with conventional chemotherapy. Feasibility has been of colonic infiltration, confirmed by colonoscopy after 10 cycles 11 demonstrated for liposomal doxorubicin. We present two of single-agent bortezomib therapy (1.3 mg/m2 intravenous, case reports and in vitro data suggesting a sequence-dependent days 1, 4, 8, 11, every 3 weeks). Four years earlier, the disease synergy of bortezomib and cytarabine in MCL. was diagnosed with colonic polyposis and infiltration of the stomach, the nasopharynx, the right lacrimal gland and bone marrow involvement. Previous treatment consisted of CHOP Case report 1 followed by interferon maintenance,12 R-FCM13 and radio- A 67-year-old man with relapsed stage IV MCL presented with immunotherapy with rituximab/90Y-ibritumomab–tiuxetan. The progressive bilateral leg edema caused by extensive inguinal, best response consisted of partial remission, and duration of iliac and paraaortic lymphadenopathy. Previous treatment response declined from initially observed 21 months with every consecutive line of treatment. Single-agent bortezomib treat- Correspondence: Professor M Dreyling, Department of Medicine III, ment resulted in a decreased frequency of diarrhea by cycle 4, University Hospital Grosshadern, Marchioninistrasse 15, D-81377 which however, failed by cycle 10. Treatment was changed to Munich, Germany. the combination regimen, consisting of bortezomib (1.5 mg/m2, E-mail: [email protected] 2 3These authors contributed equally to this work days 1 and 4) and cytarabine (1000 mg/m as a 3-h infusion on Received 21 April 2006; revised 3 October 2006; accepted 11 days 2 and 3), applied in 4-week intervals with rituximab added October 2006; published online 1 February 2007 on day 0 (375 mg/m2). Because of NCI/CTC grade 1 sensory Synergy of bortezomib and cytarabine in MCL O Weigert et al 525 Proliferation assay Cells were seeded at a density of 5 Â 105/ml in the absence or presence of bortezomib (25 nM). Cells were counted in a hematocytometer, and viability of cells was assessed by Trypan blue exclusion test (Gibco, Invitrogen, Karlsruhe, Germany) at 0, 24, 48 and 96 h of treatment.

Apoptosis assay Cells were washed and stained with Annexin V-PE/7–AAD in accordance with the manufacturer’s protocol (BD Biosciences, Heidelberg, Germany) and subjected to ﬂow cytometry (BD FACS Calibur, BD Biosciences, Palo Alto, CA, USA). Apoptosis rate was calculated as follows: 0fraction viable treated cells0 1 À Â100%: 0fraction viable untreated cells0

Spontaneous apoptosis rate was up to 10% for established MCL cell lines and up to 25% for MCL cells derived from patient samples.

Figure 1 Clinical efficacy of bortezomib and cytarabine in MCL. CT Cell viability assay and determination of Chou and scans before (a and c) and after two cycles (b and d) of combination Talalay’s combination index treatment with cytarabine and bortezomib demonstrating significant Cell viability correlates with the activity to metabolize the decrease of lymphadenopathy (arrows). tetrazolium salt WST-1 to a water-soluble formazan dye, which is measured spectrophotometrically. Cells were seeded at a density of 1 Â 106/ml in a 96-well microplate in triplicate (100 000 cells/well) and the assay was performed according to peripheral neuropathy, the dose of bortezomib was reduced to the manufacturer’s protocol (Roche Applied Science, Man- 1.0 mg/m2 during cycles 2–4. The diarrhea improved rapidly nheim, Germany). The half maximal inhibitory concentration and disappeared subsequently. After two and four cycles, a (IC ) was used for calculation of Chou and Talalay’s combina- complete disappearance of pathological lymphadenopathy was 50 tion index (CI ) applying CalcuSyn Software (Biosoft, Cam- confirmed by CT scanning (Figure 1c and d). Endoscopically, the 50 bridge, UK) as described previously.14 This index allows polypoid lesions decreased substantially however, residual MCL identification of antagonistic (CI 41.3), additive (CI ¼ infiltration was seen on biopsy specimen. A partial remission 50 50 170.3) or synergistic (CI 0.7) efficacy of combination was diagnosed and no further treatment was applied to the 50 o treatment by considering cell viability curves determined after asymptomatic patient. NCI/CTC grade 3/4 toxicity consisted 12 and 24 h of treatment with cytarabine alone, bortezomib mainly of myelotoxicity, with neutropenia requiring G-CSF alone or in combination (synchronous and sequential incuba- support and one episode of neutropenic fever requiring empiric tion) of both, respectively. antibiotic treatment. There was no treatment delay and no dose reduction of cytarabine. Meanwhile, the patient has remained in documented ongoing remission for more than 7 months (233 Statistical analysis days) after initiation of combination treatment with no disease- A two-sided Student’s t-test was applied when indicated. related symptoms.

Results Materials and methods At clinically achievable concentrations, bortezomib (25 nM) Cell culture and reagents inhibited cell growth in all cell lines in a time-dependent Established MCL cell lines consisted of Granta519, HBL-2 and manner, with MCL demonstrated to be more sensitive than the Jeko-1; the T-ALL cell line Jurkat was used as hematologic hematological control cell line Jurkat (Figure 2a, Po0.01 for control cell line; all established cell lines were obtained from MCL vs non-MCL cell lines at 48 and 96 h, Student’s t-test). DSMZ (German Collection of Microorganisms and Cell Cul- After 24 h of bortezomib treatment, IC50 assessed by cell tures, Braunschweig, Germany). After having obtained informed viability testing were 25.275.1, 22.473.1 and 14.871.6 nM for consent, primary MCL cells were collected from peripheral Granta519, HBL-2 and Jeko-1, respectively, and significantly blood or bone marrow aspiration and purified by magnetic cell lower as compared to 45.572.9 nM for Jurkat (Figure 2b, sorting for CD19 according to the manufacturer’s protocol Po0.01 for MCL vs non-MCL cell lines, Student’s t-test). (MACS, Miltenyi-Biotec, Bergisch-Gladbach, Germany). Cells Cytotoxicity of bortezomib could be enhanced by coincuba- were cultured in RPMI 1640 culture medium supplemented with tion with cytarabine, as demonstrated by assessment of cell 10–20% heat-inactivated fetal calf serum (371C, humified viability by WST-1 assay (Figure 2c) and apoptosis by flow atmosphere containing 5% carbon dioxide) and incubated with cytometry at 24 h (Figure 2d) in established MCL cell lines. The bortezomib (Velcade, Janssen-Cilag, Neuss, Germany) alone, established MCL cell lines differed remarkably concerning cytarabine (Ara-Cell, Cell Pharm, Hannover, Germany) alone, or sensitivity to cytarabine treatment (Figure 2d, white columns), in combination of both. with IC50 ranging from 1.9 to 20.4 mg/ml after 24 h as assessed

Leukemia Synergy of bortezomib and cytarabine in MCL O Weigert et al 526

Figure 2 In vitro efﬁcacy of bortezomib and cytarabine in established MCL cell lines. (a) Number of viable cells counted in a hematocytometer and stained with Trypan blue at 24, 48 and 96 h of treatment. Mean value7s.d. of three experiments. *Po0.01 (MCL vs non-MCL) from Student’s t-test. (b) Mean inhibitory concentrations (IC50) after 24 h of bortezomib treatment (WST-1 assay) of MCL cell lines (Granta519, HBL-2 and Jeko-1) and control cell line (Jurkat). Mean value7s.d. of at least three experiments. P-value (MCL vs non-MCL) from Student’s t-test. (c) Cell viability (WST-1 assay) of Jeko-1 after incubation with bortezomib, cytarabine or coincubation with both for 24 h at indicated concentrations. Mean value7s.d. of three experiments. (d) Cell apoptosis (ﬂow cytometry) of established MCL cell lines (Granta519, HBL-2 and Jeko-1) after incubation with bortezomib (25 nM, black columns), cytarabine (2 mg/ml, white columns) or both (gray columns) for 24 h. Mean value7s.d. of three experiments.

WST-1 assay (Figure 2b), this indicates that either higher concentrations or longer incubation times are required for bortezomib single-agent or combination therapy to induce cytotoxicity in the non-MCL cell line Jurkat. Strikingly, all four highly purified primary MCL samples were remarkably more sensitive to coincubation of bortezomib and cytarabine than would have been expected based on the efficacy of each monotherapy, estimated by cumulative probability (Figure 3). Impact of sequence of treatment on efficacy was investigated in established MCL cell lines by determination of CI50. Interestingly, in all MCL cell lines, preincubation with cytarabine for 12 h, followed by addition of bortezomib for another 12 h, revealed synergistic activity (CIo0.770.05–0.08) based on cell viability curves, whereas incubation conversely resulted Figure 3 In vitro efficacy of bortezomib and cytarabine in primary in antagonism in HBL-2 and Jeko-1 (Table 1). MCL cell lines. Cell apoptosis of primary MCL cells (flow cytometry) after incubation with cytarabine (1 mg/ml), bortezomib (10 nM) or both for 24 h. Cells were collected from peripheral blood or bone marrow Discussion aspiration and purified by CD19 þ MACS (495% of cells with double staining for CD5 and CD20). ‘Expected’ efficacy was calculated by applying cumulative probability based on efficacy of each mono- Our in vitro data not only confirm antilymphoma activity of therapy. bortezomib especially in MCL, but strongly suggest enhanced efficacy of bortezomib if combined with cytarabine. Interest- ingly, efficacy correlated with the sequence of treatment, by WST-1 assay, whereas sensitivity to bortezomib was indicating superiority of pretreatment with cytarabine, followed relatively stable (Figure 2d, black columns, and Figure 2b). Of by proteasome inhibition. note, bortezomib 25 nM did not induce any cytotoxic effect in Cytarabine is one of the most effective drugs used in the Jurkat cells after 24 h as assessed by flow cytometry; in line with treatment of hematologic malignancies including MCL either as the results from Trypan blue exclusion test (Figure 2a) and monotherapy15 or in combination with other cytostatic agents.16

Leukemia Synergy of bortezomib and cytarabine in MCL O Weigert et al 527 Table 1 Chou and Talalay’s combination index at IC50 (CI50) calculated based on cell viability (WST-1 assay)

Bortezomib+cytarabine Bortezomib-cytarabine Cytarabine-bortezomib

Granta 519 0.88 (70.33) 0.55 (70.21) 0.50 (70.08) HBL-2 0.79 (70.18) 2.83 (70.57) 0.17 (70.05) Jeko-1 0.64 (70.52)a 1.60 (70.81) 0.23 (70.05) Jurkat 0.40 (70.56)a 1.52 (70.23) 1.13 (70.22) Mean value7s.d. of at least three experiments. Bortezomib+cytarabine: bortezomib 24 h, cytarabine 24 h; bortezomib-cytarabine: bortezomib 24 h, cytarabine 12 h; cytarabine-bortezomib: cytarabine 24 h, bortezomib 12 h. CIo0.7 indicates synergism (bold), CI ¼ 170.3 indicates additive efﬁcacy, CI41.3 indicates antagonism. a Owing to the ﬁxed drug dose ratio by experimental design and different sensitivity of cell lines to individual drugs, estimation of the CI50 yielded a high s.d. for coincubation assays in Jeko-1 and Jurkat cells; thus, a CI50 of o0.7 does not indicate synergism in these cases.

After conversion to its triphosphate-derivative, ara–cytidine inhibits cell growth, interferes with cell cycle progression and triphosphate (CTP), it interferes with DNA polymerases and induces apoptosis in MCL. Consequently, proteasome inhibition incorporates into the genome mainly during DNA replication, before cytarabine exposure might well decrease the amount of thereby affecting the structural integrity of DNA, that is, stalling nucleoside analogue incorporation into DNA. Also, bortezo- of replication fork and chain termination. DNA damage sensors mib-induced accumulation of antiapoptotic Mcl-123 may inter- (e.g., ATM, ATR and DNA–PK) and transducers (e.g., p53, JNK, fere with efficacy of cytarabine. This may explain our CI50 Bax, Bak) activate checkpoint pathways that arrest cell cycle results: incubating MCL cells with bortezomib before cytarabine 7 progression and, if survival mechanisms are overwhelmed, yielded antagonistic efficacy in HBL-2 (CI50 ¼ 2.83 0.57) and 7 signalling pathways are activated, initiating the apoptotic Jeko-1 cells (CI50 ¼ 1.60 0.81), whereas the synergistic efficacy 17 7 pathway. Mitochondrial release of cytochrome c and other was preserved in Granta519 (CI50 ¼ 0.55 0.21), the least proapoptotic molecules (e.g., Smac/DIABLO,18 Omi/HtrA219) sensitive MCL cell line to bortezomib treatment by WST-1 assay. promotes the formation of the apoptosome, a high-molecular- Our heavily pretreated patients with relapsed advanced-stage weight-complex consisting of Apaf-1, dATP and procaspase-9. MCL had progressive lymphadenopathy after 5 and 30 weeks of Recruited caspase-9 activates effector caspases (e.g., caspase-3 single-agent bortezomib treatment. Owing to the relatively short and 7), which execute the cell death program. course of bortezomib treatment in case report 1 and the transient Considering the abundant presence and necessity of protea- response in case report 2, it remains speculative whether these some activity in eukaryotic cells, bortezomib displays surpris- lymphomas can be considered completely resistant to bortezo- ingly little toxicity in clinical practice, with mild mib. However, because of progressive clinical symptoms, a thrombocytopenia, neuropathy and diarrhea being most com- combination treatment consisting of bortezomib and cytarabine mon.5 Little is known about downstream events caused by was initiated. This particular treatment schedule was based proteasome inhibition. Gene profiling studies demonstrate primarily on clinical considerations as preliminary experience overexpression of Nuclear factor-kappa B (NF-kB)-dependent with the DA-EPOCH regimen defined the maximal-tolerated gene products in MCL, indicating constitutive activation of the dose for bortezomib (1.5 mg/m2 applied on days 1 and 4) NF-kB signalling pathway.20 NF-kB has been implicated in (Dunleavy et al., Blood 2004; 104: 389a, abstract 1385). As both blocking apoptosis, promoting cell proliferation and mediating patients developed sensory polyneuropathy, probably also treatment resistance.21 Activation of NFkB requires phosphor- because previous treatment regimens included well-known ylation of its inhibitor kinase B (IkB), leading to polyubiquiti- neurotoxic agents such as vincristine, oxaliplatin and bortezo- nylation and degradation by the proteasome. Until recently, mib itself, bortezomib had to be stopped after cycle 2 in patient antineoplastic activity of proteasome inhibitors has been largely 1, and dose was reduced in patient 2. Despite the fact that both attributed to inhibition of NF-kB activation.22 However, alter- patients never failed cytarabine-containing regimens before, this native downstream mechanisms of proteasome inhibition, such report is the first to demonstrate that combining proteasome as generation of reactive oxygen species (ROS) and upregulation inhibition with high-dose cytarabine is feasible and highly active of BH3-only mitochondrial protein Noxa, have been sug- in MCL, thus warranting further testing in clinical trials. gested.23 Noxa not only contributes to apoptosis by disrupting outer mitochondrial membrane integrity but also binds to Mcl-1,24 thereby displacing proapoptotic Bak.25 Apart from Acknowledgements Noxa and IkB, impaired proteasome activity also leads to accumulation of several other crucial cellular proteins such as This work was supported by the Friedrich-Baur-Stiftung (0078/ p21, p27, p5323 and Smac/DIABLO.19 Consequently, protea- 2005), Munich, and the European Union: 6 FP, Translational some inhibition in cells that have previously been exposed to research on promising predictive and prognostic markers: cytarabine might well further enhance chemotherapy-induced ‘European MCL Network’ (contract no. 503351) and Lymphoma increased levels of the aforementioned proapoptotic molecules, Research Foundation Grant ‘European MCL Network’. thereby promoting apoptosis. On the other hand, it has been shown that in late-stage apoptotic cells, effector caspases irreversibly inactivate proteasome activity by cleaving three References subunits of the 19S particle.19 These data suggest that the timing of proteasome inhibition may be important to augment 1 Ciechanover A. The ubiquitin–proteasome proteolytic pathway. cytotoxicity of conventional chemotherapy. Cell 1994; 79: 13–21. We (Weigert et al., Proc Am Soc Clin Oncol 2005; 23: 578s, 2 Adams J. The proteasome: a suitable antineoplastic target. Nat Rev abstract no. 6609) and others22 have shown that bortezomib Cancer 2004; 4: 349–360.

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